To broaden our knowledge on virus structure and function, a profound insight into their mechanical properties is required. Nanoindentation measurements with an atomic force microscope (AFM) are increasingly being performed to probe such material properties. This single-particle approach allows for determining the viral spring constant, their Young's modulus, as well as the force and deformation at which failure occurs. The experimental procedures for viral nanoindentation experiments are described here in detail, focusing on surface preparation, AFM imaging and nanoindentation, and the subsequent data analysis of the force-distance curves. Whereas AFM can be operated in air and in liquid, the described methods are for probing single viruses in liquid to enable working in a physiologically relevant environment. © Springer Science+Business Media, LLC 2011.